WO2001023404A1 - Isogenes cibles de medicaments: polymorphismes dans le gene d'interleukine 4 - Google Patents

Isogenes cibles de medicaments: polymorphismes dans le gene d'interleukine 4 Download PDF

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WO2001023404A1
WO2001023404A1 PCT/US2000/026608 US0026608W WO0123404A1 WO 2001023404 A1 WO2001023404 A1 WO 2001023404A1 US 0026608 W US0026608 W US 0026608W WO 0123404 A1 WO0123404 A1 WO 0123404A1
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gene
haplotype
polymoφhic
seq
individual
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PCT/US2000/026608
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English (en)
Inventor
Anne Chew
Julie Y. Choi
R. Rex Denton
Krishnan Nandabalan
Joel Claiborne Stephens
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Genaissance Pharmaceuticals, Inc.
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Priority to AU2000277254A priority Critical patent/AU2000277254A1/en
Publication of WO2001023404A1 publication Critical patent/WO2001023404A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5406—IL-4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to variation in genes that encode pharmaceutically important proteins.
  • this invention provides genetic variants of the human Interleukin-4 (IL4) gene and methods for identifying which variant(s) of this gene is/are possessed by an individual.
  • IL4 human Interleukin-4
  • a target protein currently used to screen drugs typically is expressed by a gene cloned from an individual who was arbitrarily selected.
  • the nucleotide sequence of a particular gene may vary tremendously among individuals.
  • Subtle alteration(s) in the primary nucleotide sequence of a gene encoding a target protein may be manifested as significant variation in expression of or in the structure and/or function of the protein. Such alterations may explain the relatively high degree of uncertainty inherent in treatment of individuals with drugs whose design is based upon a single representative example of the target.
  • haplotype The organization of single nucleotide variations (polymorphisms) in the primary sequence of a gene into one of the limited number of combinations that exist as units of inheritance is termed a haplotype. Each haplotype therefore contains significantly more information than individual unorganized polymorphisms. Haplotypes provide an accurate measurement of the genomic variation in the two chromosomes of an individual.
  • IL4 Interleukin-4
  • Th2 T helper type 2 cells
  • Excess IL4 production has been associated with the etiology of IgE-mediated allergies (Haas et al., supa), asthma and other atopic conditions (Song et al., J. Immunol.
  • MCNS minimal change nephrotic syndrome
  • osteoporosis Lewis et al., Proc. Natl. Acad. Sci. USA 90: 11618-11622, 1993
  • rheumatoid arthritis Rudwaleit et al. Ann Rheum Dis 2000 Apr; 59(4): 311-4.
  • Decreased IL4 production has been correlated with increasing disease activity in patients with systemic lupus erythematosus (Jones et al. Autoimmunity
  • the Interleukin-4 gene is located on chromosome 5q31.1.
  • the IL4 gene is about 10 kilobase pairs (kbp) in size and contains four exons and three introns (Arai et al., J. Immunol. 142:274-282, 1989).
  • Two alternatively-spliced transcripts of the IL4 gene have been observed; one of which contains all four exons and is translated into a precursor IL4 polypeptide of 153 amino acids (Yokoto et al, Proc Natl. Acad. Sci.
  • polymorphisms in the IL4 gene are associated with either susceptibility to atopy and other IgE-mediated conditions or to the severity of such conditions.
  • T allele of a C or T single nucleotide polymorphism (SNP) at position -590 in the IL4 gene promoter (Borish et al., Hum. Mol. Genet. 4:974, 1995) is associated with increased IL4 gene transcriptional activity and with elevated levels of total serum IgE (Rosenwasser et al., Clin. Exp. Allergy 25:74-78, 1995).
  • the occurrence of the T allele of this polymorphism is significantly higher in subjects with fatal or near-fatal asthma compared to nonasthmatic subjects (Sandford et al. J Allergy Clin Immunol 2000 Jul;106(l Pt l): 135-40).
  • Another SNP of C or T has been identified in the IL4 gene promoter at -34 (Takabayashi et al., J. Hum. Genet. 44:352-353, 1999), which probably corresponds to position 1138 of Figure 1.
  • individuals homozygous for the T allele at this position were also homozygous for the T allele at -590 (Takabayshi et al., supra).
  • a dinucleotide repeat polymorphism tightly linked to the -590 promoter SNP has also been identified in intron 2 of the IL4 gene (Dizier et al., Genet. Epidem. 16:84-94, 1999).
  • a polymorphism composed of a variable number tandem repeats (VNTR) of a 70-bp sequence which is located in the third intron may be associated with rheumatoid arthritis (Cantagrel et al., Arthritis & Rheumatism et al, 42:1093-1100 (1999).
  • IL4 gene Because of the potential for polymorphisms in the IL4 gene to affect the expression and function of the encoded protein, it would be useful to determine whether additional polymorphisms exist in the IL4 gene, as well as how such polymorphisms are combined in different copies of the gene. Such information would be useful for studying the biological function of IL4 as well as in identifying drugs targeting this protein for the treatment of disorders related to its abnormal expression or function.
  • PS polymorphic sites
  • the polymorphisms at these sites are adenine or guanine at PS2, adenine or guanine at PS3, cytosine or guanine at PS4, guanine or adenine at PS5, guanine or adenine at PS6, adenine or guanine at PS7, guanine or adenine at PS8, and cytosine or adenine at PS9.
  • the inventors have determined the identity of the alternative nucleotides present at these sites, as well as at the previously identified site at nucleotide 1138 (PSI), in a human reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: African descent, Asian, Caucasian and Hispanic/Latino. It is believed that IL4- encoding polynucleotides containing one or more of the novel polymorphic sites reported herein will be useful in studying the expression and biological function of IL4, as well as in developing drugs targeting this protein. In addition, information on the combinations of polymorphisms in the IL4 gene may have diagnostic and forensic applications.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymo ⁇ hic variant of a reference sequence for the IL4 gene or a fragment thereof.
  • the reference sequence comprises SEQ ID NO: 1 and the polymo ⁇ hic variant comprises at least one polymo ⁇ hism selected from the group consisting of guanine at PS2, guanine at PS3, guanine at PS4, adenine at PS5, adenine at PS6, guanine at PS7, adenine at PS8, and adenine at PS9.
  • the polymo ⁇ hic variant comprises an additional polymo ⁇ hism of thymine at PS 1.
  • a particularly preferred polymo ⁇ hic variant is a naturally-occurring isoform (also referred to herein as an "isogene") of the IL4 gene.
  • An IL4 isogene of the invention comprises cytosine or thymine at PSI, adenine or guanine at PS2, adenine or guanine at PS3, cytosine or guanine at PS4, guanine or adenine at PS5, guanine or adenine at PS6, adenine or guanine at PS7, guanine or adenine at PS8, and cytosine or adenine at PS9.
  • the invention also provides a collection of IL4 isogenes, referred to herein as an IL4 genome anthology.
  • An IL4 isogene may be defined by the combination and order of these polymo ⁇ hisms in the isogene, which is referred to herein as an IL4 haplotype.
  • the invention also provides data on the number of different IL4 haplotypes found in the above four population groups. This haplotype data is useful in methods for deriving an IL4 haplotype from an individual's genotype for the IL4 gene and for determining an association between an IL4 haplotype and a particular trait.
  • the invention provides a polynucleotide comprising a polymo ⁇ hic variant of a reference sequence for an IL4 cDNA or a fragment thereof.
  • the reference sequence comprises SEQ ID NO:2 (Fig. 2) and the polymo ⁇ hic cDNA comprises at least one polymo ⁇ hism selected from the group consisting of guanine at a position corresponding to nucleotide 45 and adenine at a position corresponding to nucleotide 255.
  • Polynucleotides complementary to these IL4 genomic and cDNA variants are also provided by the invention.
  • the invention provides a recombinant expression vector comprising one of the polymo ⁇ hic genomic variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector.
  • the recombinant vector and host cell may be used to express IL4 for protein structure analysis and drug binding studies.
  • the invention provides methods, compositions, and kits for haplotyping and/or genotyping the IL4 gene in an individual.
  • the methods involve identifying the nucleotide or nucleotide pair present at one or more polymo ⁇ hic sites selected from PS2, PS3, PS4, PS5, PS6, PS7, PS8, and PS9 in one or both copies of the IL4 gene from the individual.
  • the compositions contain oligonucleotide probes and primers designed to specifically hybridize to one or more target regions containing, or that are adjacent to, a polymo ⁇ hic site.
  • the methods and compositions for establishing the genotype or haplotype of an individual at the novel polymo ⁇ hic sites described herein are useful for studying the effect of the polymo ⁇ hisms in the etiology of diseases affected by the expression and function of the IL4 protein, studying the efficacy of drugs targeting IL4, predicting individual susceptibility to diseases affected by the expression and function of the IL4 protein and predicting individual responsiveness to drugs targeting IL4.
  • the invention provides a method for identifying an association between a genotype or haplotype and a trait.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • Such methods have applicability in developing diagnostic tests and therapeutic treatments for inflammatory and immune disorders.
  • the present invention also provides transgenic animals comprising one of the IL4 genomic polymo ⁇ hic variants described herein and methods for producing such animals.
  • the transgenic animals are useful for studying expression of the IL4 isogenes in vivo, for in vivo screening and testing of drugs targeted against IL4 protein, and for testing the efficacy of therapeutic agents and compounds for inflammatory and immune disorders in a biological system.
  • the present invention also provides a computer system for storing and displaying polymo ⁇ hism data determined for the IL4 gene.
  • the computer system comprises a computer processing unit; a display; and a database containing the polymo ⁇ hism data.
  • the polymo ⁇ hism data includes the polymo ⁇ hisms, the genotypes and the haplotypes identified for the IL4 gene in a reference population.
  • the computer system is capable of producing a display showing IL4 haplotypes organized according to their evolutionary relationships.
  • Figure 1 illustrates a reference sequence for the IL4 gene (Genbank Version Number M23442.1; contiguous lines; SEQ ID NO: l), with the start and stop positions of each region of coding sequence indicated below the sequence by the numbers within the brackets and the polymo ⁇ hic sites and polymo ⁇ hisms identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymo ⁇ hic site in the sequence.
  • Figure 2 illustrates a reference sequence for the IL4 coding sequence (contiguous lines; SEQ ID NO:2), with the polymo ⁇ hic sites and polymo ⁇ hisms identified by Applicants in a reference population indicated by the variant nucleotide positioned below the polymo ⁇ hic site in the sequence.
  • Figure 3 illustrates a reference sequence for the IL4 protein (contiguous lines; SEQ ID NO:3).
  • the present invention is based on the discovery of novel variants of the IL4 gene.
  • the inventors herein discovered 8 novel polymo ⁇ hic sites by characterizing the IL4 gene found in genomic DNAs isolated from Index Repository IA that contains immortalized cell lines from one chimpanzee and 93 human individuals and Index Repository IB that contains 70 human individuals. These two repositories contain 10 individuals in common.
  • Index Repository IA included a reference population of 79 unrelated individuals self-identified as belonging to one of four major population groups: Caucasian (22 individuals), African descent (20 individuals) Asian (20 individuals) Hispanic Latino (17 individuals). To the extent possible, the members of this reference population were organized into population subgroups by the self-identified ethnogeographic origin of their four grandparents as shown in Table 1 below. In addition, Index Repository IA contains three unrelated indigenous American Indians (one from each of North, Central and South America), one three-generation Caucasian family (from the CEPH Utah cohort) and one two-generation African-American family.
  • Index Repository IB contains a reference population of 70 human individuals comprised of 4 three-generation families (from the CEPH Utah cohort) as well as unrelated African- American, Asian and Caucasian individuals. A total of 38 individuals in this reference population are unrelated.
  • the IL4 genotypes identified in Index Repository 1 A include those shown in Tables 3 and 4, respectively.
  • the polymo ⁇ hism and haplotype data disclosed herein are useful for studying population diversity, anthropological lineage, the significance of diversity and lineage at the phenotypic level, paternity testing, forensic applications, and for identifying associations between the IL4 genetic variation and a trait such as level of drug response or susceptibility to disease.
  • the following terms shall be defined as follows unless otherwise indicated:
  • Allele - A particular form of a genetic locus, distinguished from other forms by its particular nucleotide sequence.
  • Candidate Gene - A gene which is hypothesized to be responsible for a disease, condition, or the response to a treatment, or to be correlated with one of these.
  • Genotype An unphased 5 ' to 3 ' sequence of nucleotide pair(s) found at one or more polymo ⁇ hic sites in a locus on a pair of homologous chromosomes in an individual.
  • genotype includes a full-genotype and/or a sub-genotype as described below.
  • Sub-genotype The unphased 5' to 3' sequence of nucleotides seen at a subset of the known polymo ⁇ hic sites in a locus on a pair of homologous chromosomes in a single individual.
  • Genotyping A process for determining a genotype of an individual.
  • Haplotype - A 5 ' to 3 ' sequence of nucleotides found at one or more polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
  • haplotype includes a full- haplotype and/or a sub-haplotype as described below.
  • Full-haplotype The 5 ' to 3 ' sequence of nucleotides found at all known polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
  • Sub-haplotype The 5' to 3' sequence of nucleotides seen at a subset of the known polymo ⁇ hic sites in a locus on a single chromosome from a single individual.
  • Haplotype pair The two haplotypes found for a locus in a single individual.
  • Haplotyping A process for determining one or more haplotypes in an individual and includes use of family pedigrees, molecular techniques and/or statistical inference.
  • Haplotype data Information concerning one or more of the following for a specific gene: a listing of the haplotype pairs in each individual in a population; a listing of the different haplotypes in a population; frequency of each haplotype in that or other populations, and any known associations between one or more haplotypes and a trait.
  • Isoform - A particular form of a gene, mRNA, cDNA or the protein encoded thereby, distinguished from other forms by its particular sequence and/or structure.
  • Isogene - One of the isoforms of a gene found in a population. An isogene contains all of the polymo ⁇ hisms present in the particular isoform of the gene.
  • Isolated - As applied to a biological molecule such as RNA, DNA, oligonucleotide, or protein, isolated means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention.
  • Locus - A location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.
  • Naturally-occurring A term used to designate that the object it is applied to, e.g., naturally- occurring polynucleotide or polypeptide, can be isolated from a source in nature and which has not been intentionally modified by man.
  • Nucleotide pair The nucleotides found at a polymo ⁇ hic site on the two copies of a chromosome from an individual.
  • phased As applied to a sequence of nucleotide pairs for two or more polymo ⁇ hic sites in a locus, phased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy of the locus is known.
  • Polymorphic site (PS) - A position within a locus at which at least two alternative sequences are found in a population, the most frequent of which has a frequency of no more than 99%.
  • Polymorphism The sequence variation observed in an individual at a polymo ⁇ hic site.
  • Polymo ⁇ hisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but need not, result in detectable differences in gene expression or protein function.
  • Polymorphism data Information concerning one or more of the following for a specific gene: location of polymo ⁇ hic sites; sequence variation at those sites; frequency of polymo ⁇ hisms in one or more populations; the different genotypes and/or haplotypes determined for the gene; frequency of one or more of these genotypes and/or haplotypes in one or more populations; any known association(s) between a trait and a genotype or a haplotype for the gene.
  • Polymorphism Database A collection of polymo ⁇ hism data arranged in a systematic or methodical way and capable of being individually accessed by electronic or other means.
  • Polynucleotide - A nucleic acid molecule comprised of single-stranded RNA or DNA or comprised of complementary, double-stranded DNA.
  • Population Group A group of individuals sharing a common ethnogeographic origin.
  • Reference Population A group of subjects or individuals who are predicted to be representative of the genetic variation found in the general population. Typically, the reference population represents the genetic variation in the population at a certainty level of at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.
  • Single Nucleotide Polymorphism SNP - Typically, the specific pair of nucleotides observed at a single polymo ⁇ hic site. In rare cases, three or four nucleotides may be found.
  • Subject A human individual whose genotypes or haplotypes or response to treatment or disease state are to be determined.
  • Treatment A stimulus administered internally or externally to a subject.
  • Unphased As applied to a sequence of nucleotide pairs for two or more polymo ⁇ hic sites in a locus, unphased means the combination of nucleotides present at those polymo ⁇ hic sites on a single copy of the locus is not known.
  • the inventors herein have discovered 8 novel polymo ⁇ hic sites in the IL4 gene.
  • the polymo ⁇ hic sites identified by the inventors are referred to as PS 1-9 to designate the order in which they are located in the gene (see Table 2 below), with the novel polymo ⁇ hic sites referred to as PS2, PS3, PS4, PS5, PS6, PS7, PS8, and PS9.
  • the invention provides an isolated polynucleotide comprising a polymo ⁇ hic variant of the IL4 gene or a fragment of the gene which contains at least one of the novel polymo ⁇ hic sites described herein.
  • the nucleotide sequence of a variant IL4 gene is identical to the reference genomic sequence for those portions of the gene examined, as described in the Examples below, except that it comprises a different nucleotide at one or more of the novel polymo ⁇ hic sites PS2, PS3, PS4, PS5, PS6, PS7, PS8, and PS9, and may also comprise an additional polymo ⁇ hism of thymine at PSI.
  • nucleotide sequence of a variant fragment of the IL4 gene is identical to the corresponding portion of the reference sequence except for having a different nucleotide at one or more of the novel polymo ⁇ hic sites described herein.
  • the invention specifically does not include polynucleotides comprising a nucleotide sequence identical to the reference sequence (or other reported IL4 sequences) or to portions of the reference sequence (or other reported IL4 sequences), except for genotyping oligonucleotides as described below.
  • polymo ⁇ hism in a variant gene or fragment is identified by aligning its sequence against SEQ ID NO:l.
  • the polymo ⁇ hism is selected from the group consisting of guanine at PS2, guanine at PS3, guanine at PS4, adenine at PS5, adenine at PS6, guanine at PS7, adenine at PS8, and adenine at PS9.
  • the polymo ⁇ hic variant comprises a naturally- occurring isogene of the IL4 gene which is defined by any one of haplotypes 1-7 shown in Table 4 below.
  • Polymo ⁇ hic variants of the invention may be prepared by isolating a clone containing the IL4 gene from a human genomic library.
  • the clone may be sequenced to determine the identity of the nucleotides at the polymo ⁇ hic sites described herein. Any particular variant claimed herein could be prepared from this clone by performing in vitro mutagenesis using procedures well-known in the art.
  • IL4 isogenes may be isolated using any method that allows separation of the two "copies" of the IL4 gene present in an individual, which, as readily understood by the skilled artisan, may be the same allele or different alleles.
  • Separation methods include targeted in vivo cloning (TIVC) in yeast as described in WO 98/01573, U.S. Patent No. 5,866,404, and U.S. Patent No. 5,972,614.
  • Another method which is described in U.S. Patent No. 5,972,614, uses an allele specific oligonucleotide in combination with primer extension and exonuclease degradation to generate hemizygous DNA targets.
  • Yet other methods are single molecule dilution (SMD) as described in Ruano et al., Proc. Natl. Acad. Sci. 87:6296-6300, 1990; and allele specific PCR (Ruano et al., 17 Nucleic Acids. Res. 8392, 1989; Ruano et al., 19 Nucleic Acids Res. 6877-6882, 1991; Michalatos-Beloin et al., 24 Nucleic Acids Res. 4841-4843, 1996).
  • the invention also provides IL4 genome anthologies, which are collections of IL4 isogenes found in a given population.
  • the population may be any group of at least two individuals, including but not limited to a reference population, a population group, a family population, a clinical population, and a same sex population.
  • An IL4 genome anthology may comprise individual IL4 isogenes stored in separate containers such as microtest tubes, separate wells of a microtitre plate and the like. Alternatively, two or more groups of the IL4 isogenes in the anthology may be stored in separate containers.
  • a preferred IL4 genome anthology of the invention comprises a set of isogenes defined by the haplotypes shown in Table 4 below.
  • An isolated polynucleotide containing a polymo ⁇ hic variant nucleotide sequence of the invention may be operably linked to one or more expression regulatory elements in a recombinant expression vector capable of being propagated and expressing the encoded IL4 protein in a prokaryotic or a eukaryotic host cell.
  • expression regulatory elements which may be used include, but are not limited to, the lac system, operator and promoter regions of phage lambda, yeast promoters, and promoters derived from vaccinia virus, adenovirus, retroviruses, or SV40.
  • regulatory elements include, but are not limited to, appropriate leader sequences, termination codons, polyadenylation signals, and other sequences required for the appropriate transcription and subsequent translation of the nucleic acid sequence in a given host cell.
  • the expression vector contains any additional elements necessary for its transfer to and subsequent replication in the host cell. Examples of such elements include, but are not limited to, origins of replication and selectable markers.
  • Such expression vectors are commercially available or are readily constructed using methods known to those in the art (e.g., F. Ausubel et al., 1987, in "Current Protocols in Molecular Biology", John Wiley and Sons, New York, New York).
  • Host cells which may be used to express the variant IL4 sequences of the invention include, but are not limited to, eukaryotic and mammalian cells, such as animal, plant, insect and yeast cells, and prokaryotic cells, such as E. coli, or algal cells as known in the art.
  • the recombinant expression vector may be introduced into the host cell using any method known to those in the art including, but not limited to, microinjection, electroporation, particle bombardment, transduction, and transfection using DEAE-dextran, lipofection, or calcium phosphate (see e.g., Sambrook et al. (1989) in "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York).
  • eukaryotic expression vectors that function in eukaryotic cells, and preferably mammalian cells, are used.
  • Non-limiting examples of such vectors include vaccinia virus vectors, adenovirus vectors, he ⁇ es virus vectors, and baculovirus transfer vectors.
  • Preferred eukaryotic cell lines include COS cells, CHO cells, HeLa cells, NIH/3T3 cells, and embryonic stem cells (Thomson, J. A. et al., 1998 Science 282: 1145-1147).
  • Particularly preferred host cells are mammalian cells.
  • polymo ⁇ hic variants of the IL4 gene will produce IL4 mRNAs varying from each other at any polymo ⁇ hic site retained in the spliced and processed mRNA molecules.
  • mRNAs can be used for the preparation of an IL4 cDNA comprising a nucleotide sequence which is a polymo ⁇ hic variant of the IL4 reference coding sequence shown in Figure 2.
  • the invention also provides IL4 mRNAs and corresponding cDNAs which comprise a nucleotide sequence that is identical to SEQ ID NO:2 (Fig.
  • polymo ⁇ hisms selected from the group consisting of guanine at a position corresponding to nucleotide 45 and adenine at a position corresponding to nucleotide 255. Fragments of these variant mRNAs and cDNAs are included in the scope of the invention, provided they contain the novel polymo ⁇ hisms described herein.
  • the invention specifically excludes polynucleotides identical to previously identified and characterized IL4 cDNAs and fragments thereof.
  • Polynucleotides comprising a variant RNA or DNA sequence may be isolated from a biological sample using well-known molecular biological procedures or may be chemically synthesized.
  • Polymo ⁇ hic variants of fragments according to the invention comprise at least one novel polymo ⁇ hism identified herein and have a length of at least 10 nucleotides and may range up to the full length of the gene.
  • such fragments are between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length.
  • nucleic acid molecules containing the IL4 gene may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand.
  • reference may be made to the same polymo ⁇ hic site on either strand and an oligonucleotide may be designed to hybridize specifically to either strand at a target region containing the polymo ⁇ hic site.
  • the invention also includes single-stranded polynucleotides which are complementary to the sense strand of the IL4 genomic variants described herein. Polynucleotides comprising a polymo ⁇ hic gene variant or fragment may be useful for therapeutic pu ⁇ oses.
  • an expression vector encoding the isoform may be administered to the patient.
  • the patient may be one who lacks the IL4 isogene encoding that isoform or may already have at least one copy of that isogene.
  • IL4 isogene In other situations, it may be desirable to decrease or block expression of a particular IL4 isogene.
  • Expression of an IL4 isogene may be turned off by transforming a targeted organ, tissue or cell population with an expression vector that expresses high levels of untranslatable mRNA for the isogene.
  • oligonucleotides directed against the regulatory regions (e.g., promoter, introns, enhancers, 3' untranslated region) of the isogene may block transcription. Oligonucleotides targeting the transcription initiation site, e.g., between positions -10 and +10 from the start site are preferred.
  • inhibition of transcription can be achieved using oligonucleotides that base-pair with region(s) of the isogene DNA to form triplex DNA (see e.g., Gee et al. in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994).
  • Antisense oligonucleotides may also be designed to block translation of IL4 mRNA transcribed from a particular isogene. It is also contemplated that ribozymes may be designed that can catalyze the specific cleavage of IL4 mRNA transcribed from a particular isogene.
  • the oligonucleotides may be delivered to a target cell or tissue by expression from a vector introduced into the cell or tissue in vivo or ex vivo.
  • the oligonucleotides may be formulated as a pharmaceutical composition for administration to the patient.
  • Oligoribonucleotides and/or oligodeoxynucleotides intended for use as antisense oligonucleotides may be modified to increase stability and half-life.
  • Possible modifications include, but are not limited to phosphorothioate or 2' O-methyl linkages, and the inclusion of nontraditional bases such as inosine and queosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytosine, guanine, thymine, and uracil which are not as easily recognized by endogenous nucleases.
  • Effect(s) of the polymo ⁇ hisms identified herein on expression of IL4 may be investigated by preparing recombinant cells and/or organisms, preferably recombinant animals, containing a polymo ⁇ hic variant of the IL4 gene.
  • expression includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into IL4 protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • the desired IL4 isogene may be introduced into the cell in a vector such that the isogene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
  • the IL4 isogene is introduced into a cell in such a way that it recombines with the endogenous IL4 gene present in the cell. Such recombination requires the occurrence of a double recombination event, thereby resulting in the desired IL4 gene polymo ⁇ hism.
  • Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention.
  • IL4 isogene
  • examples of cells into which the IL4 isogene may be introduced include, but are not limited to, continuous culture cells, such as COS, NIH/3T3, and primary or culture cells of the relevant tissue type, i.e., they express the IL4 isogene.
  • Such recombinant cells can be used to compare the biological activities of the different protein variants.
  • Recombinant organisms, i.e., transgenic animals, expressing a variant IL4 gene are prepared using standard procedures known in the art.
  • a construct comprising the variant gene is introduced into a nonhuman animal or an ancestor of the animal at an embryonic stage, i.e., the one- cell stage, or generally not later than about the eight-cell stage.
  • Transgenic animals carrying the constructs of the invention can be made by several methods known to those having skill in the art.
  • One method involves transfecting into the embryo a retrovirus constructed to contain one or more insulator elements, a gene or genes of interest, and other components known to those skilled in the art to provide a complete shuttle vector harboring the insulated gene(s) as a transgene, see e.g., U.S. Patent No. 5,610,053.
  • Another method involves directly injecting a transgene into the embryo.
  • a third method involves the use of embryonic stem cells.
  • animals into which the IL4 isogenes may be introduced include, but are not limited to, mice, rats, other rodents, and nonhuman primates (see “The Introduction of Foreign Genes into Mice” and the cited references therein, In: Recombinant DNA, Eds. J.D. Watson, M. Gilman, J. Witkowski, and M. Zoller; W.H. Freeman and Company, New York, pages 254-272).
  • Transgenic animals stably expressing a human IL4 isogene and producing human IL4 protein can be used as biological models for studying diseases related to abnormal IL4 expression and/or activity, and for screening and assaying various candidate drugs, compounds, and treatment regimens to reduce the symptoms or effects of these diseases.
  • compositions for treating disorders affected by expression or function of a novel IL4 isogene described herein.
  • the pharmaceutical composition may comprise any of the following active ingredients: a polynucleotide comprising one of these novel IL4 isogenes; an antisense oligonucleotide directed against one of the novel IL4 isogenes, a polynucleotide encoding such an antisense oligonucleotide, or another compound which inhibits expression of a novel IL4 isogene described herein.
  • the composition contains the active ingredient in a therapeutically effective amount.
  • composition also comprises a pharmaceutically acceptable carrier, examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • a pharmaceutically acceptable carrier examples of which include, but are not limited to, saline, buffered saline, dextrose, and water.
  • Those skilled in the art may employ a formulation most suitable for the active ingredient, whether it is a polynucleotide, oligonucleotide, protein, peptide or small molecule antagonist.
  • the pharmaceutical composition may be administered alone or in combination with at least one other agent, such as a stabilizing compound.
  • Administration of the pharmaceutical composition may be by any number of routes including, but not limited to oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co.,-Easton, PA).
  • the dose can be estimated initially either in cell culture assays or in animal models.
  • the animal model may also be used to determine the appropriate concentration range and route of administration.
  • Such information can then be used to determine useful doses and routes for administration in humans.
  • the exact dosage will be determined by the practitioner, in light of factors relating to the patient requiring treatment, including but not limited to severity of the disease state, general health, age, weight and gender of the patient, diet, time and frequency of administration, other drugs being taken by the patient, and tolerance/response to the treatment.
  • the invention also provides compositions and methods for detecting the novel IL4 polymo ⁇ hisms identified herein.
  • compositions comprise at least one IL4 genotyping oligonucleotide.
  • an IL4 genotyping oligonucleotide is a probe or primer capable of hybridizing to a target region that is located close to, or that contains, one of the novel polymo ⁇ hic sites described herein.
  • the term "oligonucleotide” refers to a polynucleotide molecule having less than about 100 nucleotides.
  • a preferred oligonucleotide of the invention is 10 to 35 nucleotides long. More preferably, the oligonucleotide is between 15 and 30, and most preferably, between 20 and 25 nucleotides in length.
  • oligonucleotide may be comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives.
  • oligonucleotides may have a phosphate-free backbone, which may be comprised of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and the like (Varma, R. in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Ed. R. Meyers, VCH Publishers, Inc. (1995), pages 617-620).
  • Oligonucleotides of the invention may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion.
  • the oligonucleotides may be labeled, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.
  • Genotyping oligonucleotides of the invention must be capable of specifically hybridizing to a target region of an IL4 polynucleotide, i.e., an IL4 isogene.
  • specific hybridization means the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure when incubated with a non-target region or a non-IL4 polynucleotide under the same hybridizing conditions.
  • the oligonucleotide specifically hybridizes to the target region under conventional high stringency conditions.
  • oligonucleotide probes and primers suitable for detecting polymo ⁇ hisms in the IL4 gene using the polymo ⁇ hism information provided herein in conjunction with the known sequence information for the IL4 gene and routine techniques.
  • a nucleic acid molecule such as an oligonucleotide or polynucleotide is said to be a "perfect” or “complete” complement of another nucleic acid molecule if every nucleotide of one of the molecules is complementary to the nucleotide at the corresponding position of the other molecule.
  • a nucleic acid molecule is "substantially complementary” to another molecule if it hybridizes to that molecule with sufficient stability to remain in a duplex form under conventional low-stringency conditions. Conventional hybridization conditions are described, for example, by Sambrook J. et al., in Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and by Haymes, B.D.
  • an oligonucleotide primer may have a non-complementary fragment at its 5 ' end, with the remainder of the primer being complementary to the target region.
  • non-complementary nucleotides may be interspersed into the oligonucleotide probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
  • Preferred genotyping oligonucleotides of the invention are allele-specific oligonucleotides.
  • ASO allele-specific oligonucleotide
  • allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps.
  • Allele-specific oligonucleotide probes which usually provide good discrimination between different alleles are those in which a central position of the oligonucleotide probe aligns with the polymo ⁇ hic site in the target region (e.g., approximately the 7 th or 8 th position in a 15 mer, the 8 th or 9 th position in a 16mer, the 10 th or 11 th position in a 20 mer).
  • a preferred ASO probe for detecting IL4 gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:
  • ATGAGAAGGACACTC (SEQ ID NO 12) and its complement
  • ATGAGAAAGACACTC (SEQ ID NO 13) and its complement
  • AATGTTTCTTTTGCA (SEQ ID NO 18) and its complement
  • AATGTTTATTTTGCA (SEQ ID NO 19) and its complement .
  • An allele-specific oligonucleotide primer of the invention has a 3' terminal nucleotide, or preferably a 3 ' penultimate nucleotide, that is complementary to only one nucleotide of a particular SNP, thereby acting as a primer for polymerase-mediated extension only if the allele containing that nucleotide is present. Allele-specific oligonucleotide primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • a preferred ASO primer for detecting IL4 gene polymo ⁇ hisms comprises a nucleotide sequence, listed 5 ' to 3 ', selected from the group consisting of:
  • TGTTCTTCCTGCTAG (SEQ ID NO: 20) TGCCGGCACATGCTA (SEQ ID NO: 21) TGTTCTTCCTGCTGG (SEQ ID NO 22) TGCCGGCACATGCCA (SEQ ID NO: 23) GTACCTATCTGGCAC (SEQ ID NO 24) TCTGGAGAGATGGTG (SEQ ID NO: 25) GTACCTATCTGGCGC (SEQ ID NO- 26) TCTGGAGAGATGGCG (SEQ ID NO: 27) TTCTGGTGATGCTCT (SEQ ID NO 28) CTAGAAATACTGAGA (SEQ ID NO: 29) TTCTGGTGATGCTGT (SEQ ID NO 30) CTAGAAATACTGACA (SEQ ID NO: 31) GAAGCCGTCCCACGG (SEQ ID NO 32) TGCTAAAACAGACCG (SEQ ID NO: 33) GAAGCCGTCCCACAG (SEQ ID NO 34) TGCTAAAACAGACTG (SEQ ID NO: 35) GCCACCATGAGAAGG
  • genotyping oligonucleotides of the invention hybridize to a target region located one to several nucleotides downstream of one of the novel polymo ⁇ hic sites identified herein. Such oligonucleotides are useful in polymerase-mediated primer extension methods for detecting one of the novel polymo ⁇ hisms described herein and therefore such genotyping oligonucleotides are referred to herein as "primer-extension oligonucleotides".
  • the 3 '-terminus of a primer-extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the polymo ⁇ hic site.
  • a particularly preferred oligonucleotide primer for detecting IL4 gene polymo ⁇ hisms by primer extension terminates in a nucleotide sequence, listed 5 ' to 3', selected from the group consisting of:
  • CAGGCAGGCA SEQ ID NO 62
  • AGCATTTGTC SEQ ID NO 63
  • AGGCAGGCAA SEQ ID NO 64
  • AAGCATTTGT SEQ ID NO 65
  • a composition contains two or more differently labeled genotyping oligonucleotides for simultaneously probing the identity of nucleotides at two or more polymo ⁇ hic sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymo ⁇ hic site.
  • IL4 genotyping oligonucleotides of the invention may also be immobilized on or synthesized on a solid surface such as a microchip, bead, or glass slide (see, e.g., WO 98/20020 and WO 98/20019).
  • Such immobilized genotyping oligonucleotides may be used in a variety of polymo ⁇ hism detection assays, including but not limited to probe hybridization and polymerase extension assays.
  • Immobilized IL4 genotyping oligonucleotides of the invention may comprise an ordered array of oligonucleotides designed to rapidly screen a DNA sample for polymo ⁇ hisms in multiple genes at the same time.
  • the invention provides a kit comprising at least two genotyping oligonucleotides packaged in separate containers.
  • the kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container.
  • the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as PCR.
  • the additional polymo ⁇ hic sites may be currently known polymo ⁇ hic sites or sites that are subsequently discovered.
  • One embodiment of the genotyping method involves isolating from the individual a nucleic acid mixture comprising the two copies of the IL4 gene, or a fragment thereof, that are present in the individual, and determining the identity of the nucleotide pair at one or more of the polymo ⁇ hic sites selected from PS2, PS3, PS4, PS5, PS6, PS7, PS8, and PS9 in the two copies to assign an IL4 genotype to the individual.
  • the two "copies" of a gene in an individual may be the same allele or may be different alleles.
  • the identity of the nucleotide pair at PSI is also determined.
  • the genotyping method comprises determining the identity of the nucleotide pair at each of PS 1-9.
  • the nucleic acid mixture is isolated from a biological sample taken from the individual, such as a blood sample or tissue sample.
  • tissue samples include whole blood, semen saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • the nucleic acid mixture may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from an organ in which the IL4 gene is expressed.
  • mRNA or cDNA preparations would not be used to detect polymo ⁇ hisms located in introns or in 5 ' and 3 ' nontranscribed regions. If an IL4 gene fragment is isolated, it must contain the polymo ⁇ hic site(s) to be genotyped.
  • One embodiment of the haplotyping method comprises isolating from the individual a nucleic acid molecule containing only one of the two copies of the IL4 gene, or a fragment thereof, that is present in the individual and determining in that copy the identity of the nucleotide at one or more of the polymo ⁇ hic sites PS2, PS3, PS4, PS5, PS6, PS7, PS8, and PS9 in that copy to assign an IL4 haplotype to the individual.
  • the nucleic acid may be isolated using any method capable of separating the two copies of the IL4 gene or fragment such as one of the methods described above for preparing IL4 isogenes, with targeted in vivo cloning being the preferred approach.
  • the haplotyping method also comprises identifying the nucleotide at PSI. In a particularly preferred embodiment, the nucleotide at each of PS 1-9 is identified.
  • an IL4 haplotype pair is determined for an individual by identifying the phased sequence of nucleotides at one or more of the polymo ⁇ hic sites selected from PS2, PS3, PS4, PS5, PS6, PS7, PS8, and PS9 in each copy of the IL4 gene that is present in the individual.
  • the haplotyping method comprises identifying the phased sequence of nucleotides at each of PS 1 -9 in each copy of the IL4 gene.
  • the identifying step is preferably performed with each copy of the gene being placed in separate containers.
  • first and second copies of the gene are labeled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labeled with yet a third different fluorescent dye is used to assay the polymo ⁇ hic site(s), then detecting a combination of the first and third dyes would identify the polymo ⁇ hism in the first gene copy while detecting a combination of the second and third dyes would identify the polymo ⁇ hism in the second gene copy.
  • the identity of a nucleotide (or nucleotide pair) at a polymo ⁇ hic site(s) may be determined by amplifying a target region(s) containing the polymo ⁇ hic site(s) directly from one or both copies of the IL4 gene, or fragment thereof, and the sequence of the amplified region(s) determined by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymo ⁇ hic site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site.
  • the polymo ⁇ hism may be identified directly, known as positive-type identification, or by inference, referred to as negative-type identification.
  • a site may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site.
  • the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine).
  • the identity of the allele(s) present at any of the novel polymo ⁇ hic sites described herein may be indirectly determined by genotyping a polymo ⁇ hic site not disclosed herein that is in linkage disequilibrium with the polymo ⁇ hic site that is of interest. Two sites are said to be in linkage disequilibrium if the presence of a particular variant at one site enhances the predictability of another variant at the second site (Stevens, JC 1999, Mol. Diag. 4: 309-17). Polymo ⁇ hic sites in linkage disequilibrium with the presently disclosed polymo ⁇ hic sites may be located in regions of the gene or in other genomic regions not examined herein.
  • Genotyping of a polymo ⁇ hic site in linkage disequilibrium with the novel polymo ⁇ hic sites described herein may be performed by, but is not limited to, any of the above-mentioned methods for detecting the identity of the allele at a polymo ⁇ hic site.
  • the target region(s) may be amplified using any oligonucleotide-directed amplification method, including but not limited to polymerase chain reaction (PCR) (U.S. Patent No. 4,965,188), ligase chain reaction (LCR) (Barany et al., Proc. Natl. Acad. Sci. USA 88:189-193, 1991; WO90/01069), and oligonucleotide ligation assay (OLA) (Landegren et al., Science 241:1077-1080, 1988). Oligonucleotides useful as primers or probes in such methods should specifically hybridize to a region of the nucleic acid that contains or is adjacent to the polymo ⁇ hic site.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • OLA oligonucleotide ligation assay
  • the oligonucleotides are between 10 and 35 nucleotides in length and preferably, between 15 and 30 nucleotides in length. Most preferably, the oligonucleotides are 20 to 25 nucleotides long. The exact length of the oligonucleotide will depend on many factors that are routinely considered and practiced by the skilled artisan.
  • Other known nucleic acid amplification procedures may be used to amplify the target region including transcription-based amplification systems (U.S. Patent No. 5,130,238; EP 329,822; U.S. Patent No. 5,169,766, WO89/06700) and isothermal methods (Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992).
  • a polymo ⁇ hism in the target region may also be assayed before or after amplification using one of several hybridization-based methods known in the art.
  • allele-specific oligonucleotides are utilized in performing such methods.
  • the allele-specific oligonucleotides may be used as differently labeled probe pairs, with one member of the pair showing a perfect match to one variant of a target sequence and the other member showing a perfect match to a different variant.
  • more than one polymo ⁇ hic site may be detected at once using a set of allele- specific oligonucleotides or oligonucleotide pairs.
  • the members of the set have melting temperatures within 5°C, and more preferably within 2°C, of each other when hybridizing to each of the polymo ⁇ hic sites being detected.
  • Hybridization of an allele-specific oligonucleotide to a target polynucleotide may be performed with both entities in solution, or such hybridization may be performed when either the oligonucleotide or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Allele-specific oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis.
  • Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads.
  • the solid support may be treated, coated or derivatized to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid.
  • the genotype or haplotype for the IL4 gene of an individual may also be determined by hybridization of a nucleic sample containing one or both copies of the gene to nucleic acid arrays and subarrays such as described in WO 95/11995.
  • the arrays would contain a battery of allele-specific oligonucleotides representing each of the polymo ⁇ hic sites to be included in the genotype or haplotype.
  • polymo ⁇ hisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230:1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • riboprobes Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230:1242, 1985
  • proteins which recognize nucleotide mismatches such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991).
  • variant alleles can be identified by single strand conformation polymo ⁇ hism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989).
  • SSCP single strand conformation polymo ⁇ hism
  • DGGE denaturing gradient gel electrophoresis
  • a polymerase-mediated primer extension method may also be used to identify the polymo ⁇ hism(s).
  • Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis” method (W092/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Patent 5,679,524.
  • Related methods are disclosed in WO91/02087, WO90/09455, W095/17676, U.S. Patent Nos. 5,302,509, and 5,945,283.
  • Extended primers containing a polymo ⁇ hism may be detected by mass spectrometry as described in U.S. Patent No. 5,605,798.
  • Another primer extension method is allele-specific PCR (Ruano et al., Nucl. Acids Res. 17:8392, 1989; Ruano et al., Nucl. Acids Res. 19, 6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest. 95:1635-1641, 1995).
  • multiple polymo ⁇ hic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO89/10414).
  • an individual's IL4 haplotype pair is predicted from its IL4 genotype using information on haplotype pairs known to exist in a reference population.
  • the haplotyping prediction method comprises identifying an IL4 genotype for the individual at two or more polymo ⁇ hic sites selected from PS2, PS3, PS4, PS5, PS6, PS7, PS8, and PS9, enumerating all possible haplotype pairs which are consistent with the genotype, accessing data containing IL4 haplotype pairs identified in a reference population, and assigning a haplotype pair to the individual that is consistent with the data.
  • the reference haplotype pairs include the IL4 haplotype pairs shown in Table 3.
  • the reference population should be composed of randomly-selected individuals representing the major ethnogeographic groups of the world.
  • a preferred reference population allows the detection of any haplotype whose frequency is at least 10% with about 99% certainty and comprises about 20 unrelated individuals from each of the four population groups named above.
  • a particularly preferred reference population includes a 3-generation family representing one or more of the four population groups to serve as controls for checking quality of haplotyping procedures.
  • the haplotype frequency data for each ethnogeographic group is examined to determine whether it is consistent with Hardy- Weinberg equilibrium.
  • a statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or errors in the genotyping process. If large deviations from Hardy- Weinberg equilibrium are observed in an ethnogeographic group, the number of individuals in that group can be increased to see if the deviation is due to a sampling bias. If a larger sample size does not reduce the difference between observed and expected haplotype pair frequencies, then one may wish to consider haplotyping the individual using a direct haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No.
  • the assigning step involves performing the following analysis. First, each of the possible haplotype pairs is compared to the haplotype pairs in the reference population. Generally, only one of the haplotype pairs in the reference population matches a possible haplotype pair and that pair is assigned to the individual.
  • the individual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair.
  • the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, CLASPER System TM technology (U.S. Patent No.
  • the invention also provides a method for determining the frequency of an IL4 genotype or IL4 haplotype in a population.
  • the method comprises determining the genotype or the haplotype pair for the IL4 gene that is present in each member of the population, wherein the genotype or haplotype comprises the nucleotide pair or nucleotide detected at one or more of the polymo ⁇ hic sites PS2, PS3, PS4, PS5, PS6, PS7, PS8, and PS9 in the IL4 gene; and calculating the frequency any particular genotype or haplotype is found in the population.
  • the population may be a reference population, a family population, a same sex population, a population group, a trait population (e.g., a group of individuals exhibiting a trait of interest such as a medical condition or response to a therapeutic treatment).
  • frequency data for IL4 genotypes and/or haplotypes found in a reference population are used in a method for identifying an association between a trait and an IL4 genotype or an IL4 haplotype.
  • the trait may be any detectable phenotype, including but not limited to susceptibility to a disease or response to a treatment.
  • the method involves obtaining data on the frequency of the genotype(s) or haplotype(s) of interest in a reference population as well as in a population exhibiting the trait.
  • Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one of the methods described above.
  • the haplotypes for the trait population may be determined directly or, alternatively, by the predictive genotype to haplotype approach described above.
  • the frequency data for the reference and/or trait populations is obtained by accessing previously determined frequency data, which may be in written or electronic form.
  • the frequency data may be present in a database that is accessible by a computer. Once the frequency data is obtained, the frequencies of the genotype(s) or haplotype(s) of interest in the reference and trait populations are compared. In a preferred embodiment, the frequencies of all genotypes and/or haplotypes observed in the populations are compared.
  • the trait is predicted to be associated with that IL4 genotype or haplotype.
  • the IL4 genotype or haplotype being compared in the trait and reference populations is selected from the full-genotypes and full-haplotypes shown in Tables 3 and 4, respectively, or from sub-genotypes and sub-haplotypes derived from these genotypes and haplotypes.
  • the trait of interest is a clinical response exhibited by a patient to some therapeutic treatment, for example, response to a drug targeting IL4 or response to a therapeutic treatment for a medical condition.
  • medical condition includes but is not limited to any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment is desirable, and includes previously and newly identified diseases and other disorders.
  • clinical response means any or all of the following: a quantitative measure of the response, no response, and adverse response (i.e., side effects).
  • clinical population In order to deduce a correlation between clinical response to a treatment and an IL4 genotype or haplotype, it is necessary to obtain data on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population".
  • This clinical data may be obtained by analyzing the results of a clinical trial that has already been run and/or the clinical data may be obtained by designing and carrying out one or more new clinical trials.
  • the term "clinical trial” means any research study designed to collect clinical data on responses to a particular treatment, and includes but is not limited to phase I, phase II and phase UI clinical trials. Standard methods are used to define the patient population and to enroll subjects.
  • the individuals included in the clinical population have been graded for the existence of the medical condition of interest. This is important in cases where the symptom(s) being presented by the patients can be caused by more than one underlying condition, and where treatment of the underlying conditions are not the same. An example of this would be where patients experience breathing difficulties that are due to either asthma or respiratory infections. If both sets were treated with an asthma medication, there would be a spurious group of apparent non-responders that did not actually have asthma. These people would affect the ability to detect any correlation between haplotype and treatment outcome.
  • This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use haplotyping for situations where there is a strong correlation between haplotype pair and disease susceptibility or severity.
  • the therapeutic treatment of interest is administered to each individual in the trial population and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses and that the investigator will choose the number of responder groups (e.g., low, medium, high) made up by the various responses.
  • the IL4 gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment. After both the clinical and polymo ⁇ hism data have been obtained, correlations between individual response and IL4 genotype or haplotype content are created. Correlations may be produced in several ways.
  • individuals are grouped by their IL4 genotype or haplotype (or haplotype pair) (also referred to as a polymo ⁇ hism group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymo ⁇ hism group are calculated. These results are then analyzed to determine if any observed variation in clinical response between polymo ⁇ hism groups is statistically significant.
  • haplotype or haplotype pair
  • Statistical analysis methods which may be used are described in L.D. Fisher and G. vanBelle, "Biostatistics: A Methodology for the Health Sciences", Wiley-Interscience (New York) 1993. This analysis may also include a regression calculation of which polymo ⁇ hic sites in the IL4 gene give the most significant contribution to the differences in phenotype.
  • PCT regression model useful in the invention is described in PCT
  • a second method for finding correlations between IL4 haplotype content and clinical responses uses predictive models based on error-minimizing optimization algorithms.
  • One of many possible optimization algorithms is a genetic algorithm (R. Judson, "Genetic Algorithms and Then- Uses in Chemistry” in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, K. B. Lipkowitz and D. B. Boyd, eds. (VCH Publishers, New York, 1997).
  • Simulated annealing Press et al., "Numerical Recipes in C: The Art of Scientific Computing", Cambridge University Press (Cambridge) 1992, Ch. 10), neural networks (E. Rich and K.
  • Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much of the variation in the clinical data is explained by different subsets of the polymo ⁇ hic sites in the IL4 gene.
  • ANOVA analysis of variation
  • PCT/USOO/17540 ANOVA is used to test hypotheses about whether a response variable is caused by or correlated with one or more traits or variables that can be measured (Fisher and vanBelle, supra, Ch. 10).
  • a mathematical model may be readily constructed by the skilled artisan that predicts clinical response as a function of IL4 genotype or haplotype content.
  • the model is validated in one or more follow-up clinical trials designed to test the model. The identification of an association between a clinical response and a genotype or haplotype
  • the diagnostic method may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the IL4 gene), a serological test, or a physical exam measurement.
  • a direct DNA test i.e., genotyping or haplotyping one or more of the polymo ⁇ hic sites in the IL4 gene
  • a serological test i.e., a serological test
  • a physical exam measurement i.e., a physical exam measurement. The only requirement is that there be a good correlation between the diagnostic test results and the underlying IL4 genotype or haplotype that is in turn correlated with the clinical response.
  • this diagnostic method uses the predictive haplotyping method described above.
  • Any or all analytical and mathematical operations involved in practicing the methods of the present invention may be implemented by a computer.
  • the computer may execute a program that generates views (or screens) displayed on a display device and with which the user can interact to view and analyze large amounts of information relating to the IL4 gene and its genomic variation, including chromosome location, gene structure, and gene family, gene expression data, polymo ⁇ hism data, genetic sequence data, and clinical data population data (e.g., data on ethnogeographic origin, clinical responses, genotypes, and haplotypes for one or more populations).
  • the IL4 polymo ⁇ hism data described herein may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files). These polymo ⁇ hism data may be stored on the computer's hard drive or may, for example, be stored on a CD ROM or on one or more other storage devices accessible by the computer. For example, the data may be stored on one or more databases in communication with the computer via a network.
  • a relational database e.g., an instance of an Oracle database or a set of ASCII flat files.
  • These polymo ⁇ hism data may be stored on the computer's hard drive or may, for example, be stored on a CD ROM or on one or more other storage devices accessible by the computer.
  • the data may be stored on one or more databases in communication with the computer via a network.
  • This example illustrates examination of various regions of the IL4 gene for polymo ⁇ hic sites using DNA from Index Repository 1 A.
  • Target Regions The following target regions of the IL4 gene were amplified using the PCR primer pairs listed below, with the sequences presented in the 5 ' to 3 ' direction and nucleotide positions shown for each region corresponding to the indicated GenBank Accession No.
  • Amplification profile 94°C - 2 min. 1 cycle
  • PCR products were purified by Solid Phase Reversible Immobilization using the protocol developed by the Whitehead Genome Center. A detailed protocol can be found at http://www.genome.wi.mit.edu/sequencing/protocols/pure/SPRI_pcr.html. Briefly, five ⁇ l of carboxyl coated magnetic beads (10 mg/ml) and 60 ⁇ l of HYB BUFFER (2.5M NaCl/20% PEG 8000) were added to each PCR reaction mixture (20 ⁇ l). The reaction mixture was mixed well and incubated at room temperature (RT) for 10 min. The microtitre plate was placed on a magnet for 2 min and the beads washed twice with 150 ⁇ l of 70% EtOH. The beads were air dried for 2 min and the DNA was eluted in 25 ⁇ l of distilled water and incubated at RT for 5 min. The beads were magnetically separated and the supernatant removed for testing and sequencing.
  • HYB BUFFER 2.5M NaCl/20% PEG 8000
  • the purified PCR products were sequenced in both directions using the primer sets described previously or those listed, in the 5 ' to 3 ' direction, below.
  • Fragment 4 Forward Primer 1434-1453 GAGGCTTCTCCCTGCCACTC (SEQ ID NO: 86) Reverse Primer Complement of 1915-1896 TCCAGGCACTATGCCAAAGC (SEQ ID NO:87) Fragment 5
  • This example illustrates examination of the IL4 gene for polymo ⁇ hic sites in several regions spanning about 200 base pairs upstream of the ATG start site to about 115 base pairs downstream of the termination codon. Amplification of Target Regions
  • primer pairs were used in PCR reactions containing genomic DNA isolated from immortalized cell lines for each member of a reference population of 70 human individuals.
  • the PCR reactions were carried out under the following conditions:
  • PCR products were purified by Solid Phase Reversible Immobilization using the protocol developed by the Whitehead Genome Center. A detailed protocol can be found at http://www.genome.wi.mit.edu/sequencing/protocols/pure/SPRI_pcr.html.
  • carboxyl coated magnetic beads (10 mg/ml) were washed three times with wash buffer (0.5 M EDTA, pH 8.0). Ten ⁇ l of washed beads and 50 ⁇ l of HYB BUFFER (2.5M NaCl/20% PEG 8000) were added to each PCR reaction mixture (50 ⁇ l). The reaction mixture was mixed well and incubated at room temperature (RT) for 10 min. The microtitre plate was placed on a magnet for 2 min and the beads washed twice with 150 ⁇ l of 70% EtOH. The beads were air dried for 2 min and resuspend in 20 ⁇ l of elution buffer (10 mM trisacetate, pH 7.8) and incubated at RT for 5 min. The beads were magnetically separated and the supernatant removed for testing and sequencing.
  • wash buffer 0.5 M EDTA, pH 8.0
  • Ten ⁇ l of washed beads and 50 ⁇ l of HYB BUFFER 2.5M NaCl/20% PEG 8000
  • This example illustrates analysis of the IL4 polymo ⁇ hisms identified in the Index Repositories for human genotypes and haplotypes for all polymo ⁇ hic sites except PS3, PS5, and PS 8.
  • the different genotypes containing these polymo ⁇ hisms that were observed in the reference population are shown in Table 3 below, with the haplotype pair indicating the combination of haplotypes determined for the individual using the haplotype derivation protocol described below.
  • Table 3 homozygous positions are indicated by one nucleotide and heterozygous positions are indicated by two nucleotides Missing nucleotides in any given genotype in Table 3 were inferred based on linkage disequilibrium and/or Mendelian inheritance.
  • haplotype pairs shown in Table 3 were estimated from the unphased genotypes using an extension of Clark's algorithm (Clark, A.G. (1990) Mol Bio Evol 7, 111-122), as described in U.S. Provisional Application Serial No. 60/198,340 entitled "A Method and System for Determining Haplotypes from a Collection of Polymo ⁇ hisms".
  • haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one of the variable sites.
  • the list of haplotypes was augmented with haplotypes obtained from two families (a three-generation Caucasian family and a two-generation African-American family). This list of haplotypes is then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals.

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Abstract

L'invention concerne des polynucléotides comprenant au moins un des 8 nouveaux polymorphismes de nucléotides uniques dans le gène d'interleukine 4 (IL4) humain. Cette invention concerne également des compositions et des méthodes de détection d'au moins un de ces polymorphismes, ainsi que divers génotypes et haplotypes pour le gène IL4 qui existent dans la population.
PCT/US2000/026608 1999-09-30 2000-09-28 Isogenes cibles de medicaments: polymorphismes dans le gene d'interleukine 4 WO2001023404A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1405921A1 (fr) * 2002-10-04 2004-04-07 Roche Diagnostics GmbH Détection de susceptibilité pour des maladies auto-immunes, spécifiquement le diabète de type 1
US7205106B1 (en) 2001-07-20 2007-04-17 Roche Molecular Systems, Inc. Association of polymorphisms in IL4-related genes with autoimmune disease

Citations (1)

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US5833975A (en) * 1989-03-08 1998-11-10 Virogenetics Corporation Canarypox virus expressing cytokine and/or tumor-associated antigen DNA sequence

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Publication number Priority date Publication date Assignee Title
US5833975A (en) * 1989-03-08 1998-11-10 Virogenetics Corporation Canarypox virus expressing cytokine and/or tumor-associated antigen DNA sequence

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Title
ARAI ET AL.: "Complete nucleotide sequence of the chromosomal gene for human IL-4 and its expression", J. IMMUNOLOGY, vol. 142, no. 1, 1989, pages 274 - 282, XP002937143 *
HIJAZI ET AL.: "Interleukin-4 gene promoter polymorphism (c590T) and asthma in Kuwaiti Arabs", INT. ARCH. ALLERGY IMMUNOL., vol. 122, 2000, pages 190 - 194, XP002937142 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7205106B1 (en) 2001-07-20 2007-04-17 Roche Molecular Systems, Inc. Association of polymorphisms in IL4-related genes with autoimmune disease
EP1405921A1 (fr) * 2002-10-04 2004-04-07 Roche Diagnostics GmbH Détection de susceptibilité pour des maladies auto-immunes, spécifiquement le diabète de type 1

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